Methods for synthesizing and purifying aminoalkyl tetracycline compounds

ABSTRACT

Methods for the synthesis and purification of 9-amino alkyl tetracycline compounds are described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/926,461; filed on Apr. 27, 2007, the entire contents of which arehereby incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbactericidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280;3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains, at least in part, to a methodof synthesizing an aminoalkyl tetracycline compound. The method includescontacting a tetracycline compound with an N-hydroxymethyl-phthalimidein the presence of a water scavenger and an acid under appropriateconditions, such that an aminomethyl tetracycline intermediate compoundis formed.

In another embodiment, the invention pertains, at least in part, to amethod for the synthesis of an aminoalkyl tetracycline compound. Themethod includes: contacting a tetracycline compound with aN-hydroxymethyl-phthalimide in the presence of a water scavenger and anacid under appropriate conditions to form an aminomethyl tetracyclineintermediate compound; treating the aminomethyl tetracyclineintermediate compound with methylamine under second appropriateconditions to form a second aminomethyl tetracycline intermediate; andtreating the second aminomethyl tetracycline intermediate underappropriate hydrogenation conditions, such that an aminomethyltetracycline compound is formed.

In another embodiment, the invention pertains, at least in part, to amethod of purifying alkylaminomethyl minocycline compounds by injectingan aqueous low pH solution of the compound into an HPLC in a polarorganic solvent gradient, and combining the product fractions containingthe alkylaminomethyl minocycline compound.

In yet another embodiment, the invention pertains, at least in part, toa method of removing hydrophobic impurities and oxidative degradentsfrom an alkylaminomethyl minocycline compound. The invention includesdissolving the minocycline compound in an aqueous solution of a pH of4.0-4.5, washing the aqueous solution with a non-polar organic solvent,and retaining the aqueous solution, such that hydrophobic impurities andoxidative degradents are removed from the alkylaminomethyl minocyclinecompound.

In yet another embodiment, the invention also pertains, at least inpart, to a method of removing the β epimer and by products from analkylaminomethyl minocycline compound. The method includes dissolvingthe minocycline compounds in an aqueous solution of a pH of 7.5-8.5,washing the aqueous solution with a non-polar organic solvent, andretaining the organic solution, such that hte β epimer and by productsare removed from the alkylaminomethyl minocycline compound.

In yet another embodiment, the invention includes pharmaceuticalcompositions comprising a tetracycline compound synthesized and/orpurified by the methods of the invention and a pharmaceuticallyacceptable carrier.

In yet another embodiment, the invention includes a method for treatinga tetracycline responsive state in a subject, by administering to thesubject an effective amount of a tetracycline compound synthesizedand/or purified by the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION 1. Methods for SynthesizingAmino-Methyl Tetracycline Compounds

In an embodiment, the invention pertains to an improved synthesis of4-α-9-amino methyl tetracyclines with low 4-β-epimer content.Epimerization of tetracycline compounds at the C-4 position has been achallenge for chemists working to synthesize new tetracyclinederivatives.

In one embodiment, method of synthesizing an aminoalkyl tetracyclinecompound, comprising contacting a tetracycline compound with aN-hydroxymethyl-phthalimide in the presence of an acid and a waterscavenger under appropriate conditions, such that an aminomethyltetracycline intermediate compound is formed.

The term “tetracycline compound” includes many compounds with a similarring structure to tetracycline. Examples of tetracycline compoundsinclude: tetracycline, chlortetracycline, oxytetracycline,demeclocycline, methacycline, sancycline, doxycycline, and minocycline.Other derivatives and analogues comprising a similar four ring structureare also included. Table 1 depicts tetracycline and several knowntetracycline derivatives. The C-4 position is marked by an arrow.

TABLE I

The tetracycline compound may be substituted at any position of thetetracycline ring. For example, the tetracycline compound may further besubstituted at the 1, 2, 3, 4, 5, 6, 7, 8, 10, 10a, 11, 11a, 12, 12a,and/or 13 position. The term tetracycline compound also includescompounds of the formula (I):

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkyl sulfinyl, alkyl sulfonyl,alkylamino, aryl alkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R³, R¹¹ and R¹² are each hydrogen, or a pro-drug moiety;

R¹⁰ is hydrogen, a prodrug moiety, or linked to R⁹ to form a ring;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkyl sulfonyl, alkylamino, or an aryl alkyl;

R⁷ is hydrogen, halogen, nitro, alkyl, alkenyl, heterocyclic, acyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, or—(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁹ is hydrogen;

W is CR^(7d)R^(7e), S, NR^(7b) or O;

W′ is O, NR^(7f) S;

R^(7a), R^(7b), R^(7e), R^(7d), R^(7e), and R^(7f) are eachindependently hydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl,heterocyclic, heteroaromatic or a prodrug moiety;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In a further embodiment, the tetracycline compound is minocycline. Thestructure of minocycline is shown below:

The term “N-hydroxymethyl phthalimide” includes compounds of theformula:

wherein

R¹ and R² are each hydrogen, alkyl, halogen, alkenyl, alkynyl, aryl,cyano, amino, amidino, alkoxy, and acyl. In a further embodiment, R¹ andR² are each alkyl or hydrogen.

The term “acid” includes organic acids such as triflic acid, methanesulfonic acid, and fuming sulfuric acid. Other acids known in the artare also included such as hydrochloric, sulfuric, phosphoric,hydrobromic, etc.

The term “water scavenger” includes agents which remove moisture fromthe reaction. These agents include acid chlorides and acid anhydrides(e.g., methyl sulfonic anhydride).

In certain embodiments, the reaction is conducted at a temperature ofabout 20-25° C. In certain embodiments, the reaction is stirred forabout 1-2 hours after the addition of a first portion ofN-hydroxymethylphthalimide. An additional portion ofN-hydroxyphthalimide may be added, keeping the temperature between20-35° C. The reaction may be continued until HPLC analysis of analiquot of the reaction confirms that less than 4 area percent ofmono-alkylated minocycline is remaining. The reaction conditions mayfurther comprise a work up of adding the solution to an ice chilledflask of water, where the temperature is kept below 25° C. The productmay be filtered, washed with water, and brought to a neutral pH. In afurther embodiment, the appropriate conditions include an inertatmosphere (e.g., nitrogen or argon). In addition, the aminomethyltetracycline intermediate may further be disolved in acetone oracetonitrile to effect a more precise neutralization.

In another further embodiment, the resulting aminomethyl tetracyclineintermediate comprises a 9:1 ratio of bis substituted (e.g., substitutedat the 2 and 9 position) to tris substituted (e.g., substituted at the2, 9 and 10 position). In a further embodiment, the amount of bissubstituted amino methyl tetracycline compound is greater than about50%, greater than about 60%, greater than about 70%, greater than about80%, or greater than about 90%.

In a further embodiment, the aminomethyl tetracycline intermediatecompound may be of the formula:

The aminomethyl tetracycline intermediate may be further treated withmethylamine to form an aminomethyl tetracycline compound. The treatmentwith methylamine may be performed in an alkyl alcohol (e.g., EtOH, MeOH,etc.) solvent. The phthalamide by-product which forms may be removed byprecipitation and the product may be precipitated from a mixture ofcyclic/alkyl ether and alkyl alcohol in a ratio of about 1.5:1. In afurther embodiment, the appropriate amount of the alkyl alcohol is anamount sufficient to prevent the precipitation of an aminomethyltetracycline intermediate from the reaction.

In a further embodiment, the invention pertains to a method for thesynthesis of an aminoalkyl tetracycline compound. The method includescontacting a tetracycline compound with a N-hydroxymethyl-phthalimide inthe presence of an acid and a water scavenger under appropriateconditions to form an aminomethyl tetracycline intermediate compound;treating said aminomethyl tetracycline intermediate compound withmethylamine under second appropriate conditions to form a secondaminomethyl tetracycline intermediate; treating the second aminomethyltetracycline intermediate under appropriate hydrogenation conditions,such that an amino methyltetracycline compound is formed. In a furtherembodiment, the tetracycline compound is minocycline.

In a further embodiment, the second appropriate conditions includedissolving the aminomethyl tetracycline intermediate compound in cyclicor alkyl ether and an appropriate amount of an alkyl alcohol. The alkylalcohol may be used, for example, to selectively precipitate the byproducts and the cyclic or alkyl ether may be used to selectivelyprecipitate the aminoalkyl tetracycline intermediate.

The resulting second aminomethyl tetracycline intermediate can then beisolated as a free base. Different salts of the free base can also beformed to effect a purification. Examples of acids which can be used toconvert the free base to a salt include, but are not limited to, HCl,trifluoroacetic acid, methylsulfonic acid, and acetic acid. For example,when precipitating a hydrochloride salt, hydrochloric acid inisopropanol is added to a suspension of the compound in methanol to a pHof about 3.0. The mixture is stirred and filtered if necessary to removeinsoluble constituents. The salt of the second aminomethyl tetracyclinemay be precipitated with t-butylmethylether and isolated by filtration.The salts of the second aminomethyl tetracycline intermediates may thendirectly undergo hydrogenation.

Examples of second aminomethyl tetracycline intermediates includecompounds of the formula:

In a further embodiments, the appropriate hydrogenation conditionsinclude transferring the second aminomethyl tetracycline intermediate toa hydrogenation flask and charging the flask with Pd/C or Pd/C/S eitherwet or as a dry powder. The reaction may be conducted using solvent suchas methanol and an aldehyde. Appropriate hydrogenation conditions alsoinclude the use of hydrogen gas, which may be incorporated by conductingthe reaction under a pressure of about 30 psi. The hydrogenation mayoccur for an appropriate length of time, such as about 24 hours. Anexample of an amino methyl tetracycline compound is:

In a further embodiment, the aminomethyl tetracycline compound iscontacted with an aldehyde or ketone under appropriate conditions, suchthat a substituted aminomethyl tetracycline compound is formed. Examplesof substituted aminomethyl tetracycline compounds, include compounds ofthe formula:

wherein R^(A) and R^(B) are each independently hydrogen, alkyl, alkenyl,alkynyl or aryl.

In one embodiment, R^(B) is hydrogen and R^(A) is alkyl (e.g.,(CH₃)₃CCH₂—). In another embodiment, the substituted aminomethyltetracycline compound is:

The 4-α and the 4-β epimer of an aminomethyl minocycline compound areshown below. The arrow marks the C-4 position of the tetracycline ringsystem.

The term “epimeric purity” refers to the % of the tetracycline compounds(e.g., a tetracycline compound of the invention, (e.g., a substitutedaminoalkyl tetracycline compound, an aminomethyl tetracycline compound,or an alkylaminomethyl minocycline compound) in a given sample with aparticular desired epimeric configuration. In one embodiment, theepimeric purity of a tetracycline compound of the invention is greaterthan 95% of the α-epimer of a tetracycline compound at the C-4 position.In a further embodiment, the epimeric purity of the tetracyclinecompound is at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or at least 99.9% the α-epimer at the C-4 position of thetetracycline compound.

Preferably, the substituted aminoalkyl tetracycline compound,aminomethyltetracycline compound, or alkylaminomethyl minocycline compound of theinvention comprises mostly (e.g., at least 50%) α-C-4 epimer. In afurther embodiment, the compound is about 60% α-C-4 epimer, about 70%α-C-4 epimer, about 80% α-C-4 epimer, about 90% α-C-4 epimer, at leastabout 95% α-C-4 epimer, or at least about 97% α-C-4 epimer. In a furtherembodiment, the substituted aminoalkyltetracycline compound, aminomethyltetracycline compound, or alkylaminomethyl minocycline compound of theinvention comprises less than about 7% β-C-4 epimer, less than about 5%β-C-4 epimer, or less than about 3% α-C-4 epimer.

The epimeric purity of a particular tetracycline compound made by themethods of the invention can be determined by using methods known in theart. For example, epimeric purity can be determined by HPLC or highfield NMR.

In one embodiment, any hydrophobic impurities and oxidative degradentsof the aminoalkyl tetracycline compound, aminomethyl tetracyclinecompound, or alkylaminomethyl minocycline compound of the invention(e.g., late-eluting hydrophobic impurities such as 4-carbonyl byproducts and other oxidative degradents from the acidic aqueous solutionof particular compound of the invention) may be removed by washing theaqueous solution with a non-polar organic solvent (e.g., CH₂Cl₂). Theorganic layers may be discarded and the aqueous layers may then becombined and retained.

2. Methods for the Isolation and Purification of 9-Alkyl Amino MethylTetracycline Compounds

In another embodiment, the invention pertains to methods of purifying9-alkyl amino methyl tetracyclines (or any tetracycline compound capableof being purified by the present methods) from impurities, β-epimer, andby products.

In one embodiment, the invention pertains to a method of purifyingalkylaminomethyl minocycline compounds using chromatography. The methodincludes injecting an aqueous low pH solution of the compound into anHPLC in a polar organic solvent gradient, and combining the productfractions, such that the alkylaminomethyl minocycline compound ispurified.

It has been found that selection of suitable acidic mobile phasesenhances process stability and selectivity. Organic and mineral acidmobile phases may be effective for separating by-products includingepimer impurities and closely-eluting by products through pH control orchoice of acid. Acidic mobile phases may also protect against oxidativedegradation of the minocycline compound.

In a further embodiment, the low pH solution has a pH of between about2-3. Examples of solutions that may be used include 0.1% aqueoussolutions of methane sulfonic acid. In certain embodiments, an isocraticgradient of 94% of the aqueous solution and 6% acetonitrile or anotherpolar organic solvent may be used to purify the minocycline compoundfrom epimeric and closely eluting by-products.

The resulting aqueous product fractions may be combined and the pH maybe adjusted to between about 4.0-4.5 using a base (e.g., NaOH).Hydrophobic impurities and oxidative degradents of the minocyclinecompound may be removed by washing the aqueous solution with a non-polarorganic solvent (e.g., CH₂Cl₂). The organic layers may then be discardedand the aqueous layers are combined and retained.

It should be noted that the organic solvents, such as methylenechloride, may be used to selectively remove late-eluting hydrophobicimpurities such as 4-carbonyl by products and other oxidative degradentsfrom the acidic aqueous solution of the minocycline compound.

The pH of the combined aqueous layers may then be adjusted to neutralpH, e.g., about 7.5 to about 8.5. The pH may be adjusted by the additionof a base, such as NaOH. The neutral solution is then washed with anon-polar organic solvent, such as methylene chloride. It should benoted that selective pH adjustment to neutral pH ranges also allows theminocycline compound to be extracted into the organic solvent whileretaining undesired β-epimer and by products are dissolved the aqueousphase.

In addition, antioxidants may also be added to the aqueous solutions ofminocycline compounds described herein. The antioxidants may be providedto prevent oxidative degradation of the minocycline compounds. Suitableantioxidants include, for example, sulfites (e.g., meta bisulfite,bisulfite, ammonium sulfite, etc.), citric acid, etc.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic)groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone. In certainembodiments, a straight chain or branched chain alkyl has 20 or fewercarbon atoms in its backbone (e.g., C₁-C₂₀ for straight chain, C₃-C₂₀for branched chain), and more preferably 4 or fewer. Cycloalkyls mayhave from 3-8 carbon atoms in their ring structure, and more preferablyhave 5 or 6 carbons in the ring structure. The term C₁-C₆ includes alkylgroups containing 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkyl carbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonyl amino, arylcarbonylamino, carbamoyl andureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can alsobe fused or bridged with alicyclic or heterocyclic rings which are notaromatic so as to form a polycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 20 or fewer carbon atoms inits backbone (e.g., C₂-C₂₀ for straight chain, C₃-C₂₀ for branchedchain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms intheir ring structure, and more preferably have 5 or 6 carbons in thering structure. The term C₂-C₂₀ includes alkenyl groups containing 2 to20 carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkyl arylamino),acylamino (including alkylcarbonyl amino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,non ynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 20 or fewer carbon atoms in its backbone (e.g., C₂-C₂₀for straight chain, C₃-C₂₀ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including, e.g., alkylcarbonylamino, arylcarbonylamino,carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkenyl, alkynyl groups,halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryl oxycarbonyloxy, carboxylate, alkyl carbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “amide” or “aminocarboxy” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms “alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻X⁺,where X⁺ is a counter ion.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety,

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

3. Pharmaceutical Compositions Comprising Tetracycline Compounds of theInvention

In a further embodiment, the invention pertains to pharmaceuticalcompositions comprising a tetracycline compound of the invention (e.g.,synthesized, or purified by the methods of the invention) or apharmaceutically acceptable salt, prodrug or ester thereof. Thepharmaceutical compositions may comprise a pharmaceutically acceptablecarrier.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform may be accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 1.0 to about 100 mg per kg per day. If desired,the effective daily dose of the active compound may be administered astwo, three, four, five, six or more sub-doses administered separately atappropriate intervals throughout the day, optionally, in unit dosageforms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

As set out above, certain embodiments of the present compounds cancontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” is art recognized and includes relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Farm. SCI.66:1-19).

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesincludes relatively non-toxic, inorganic and organic base addition saltsof compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. Hydroxyls canbe converted into esters via treatment with an esterifying agent such asalkanoyl halides. The term also includes lower hydrocarbon groupscapable of being solvated under physiological conditions, e.g., alkylesters, methyl, ethyl and propyl esters. (See, for example, Berge etal., supra.).

The invention also pertains to tetracycline compounds, which aresynthesized and/or purified by the methods of the invention, andpharmaceutically acceptable salts thereof.

4. Methods of Using the Tetracycline Compounds of the Invention

The invention also pertains to a method for treating a tetracyclineresponsive state in a subject, by administering to the subject aneffective amount of a tetracycline compound synthesized and/or purifiedby the method the invention or a pharmaceutically acceptable saltthereof, such that the state is treated.

The term “treating” includes curing as well as ameliorating at least onesymptom of the state, disease or disorder, e.g., the tetracyclinecompound responsive state.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention. Tetracycline compound responsive states includebacterial, viral, and fungal infections (including those which areresistant to other tetracycline compounds), cancer (e.g., prostate,breast, colon, lung melanoma and lymph cancers and other disorderscharacterized by unwanted cellular proliferation, including, but notlimited to, those described in U.S. Pat. No. 6,100,248), arthritis,osteoporosis, diabetes, and other states for which tetracyclinecompounds have been found to be active (see, for example, U.S. Pat. Nos.5,789,395; 5,834,450; 6,277,061 and 5,532,227, each of which isexpressly incorporated herein by reference). Compounds of the inventioncan be used to prevent or control important mammalian and veterinarydiseases such as diarrhea, urinary tract infections, infections of skinand skin structure, ear, nose and throat infections, wound infection,mastitis and the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)). In a furtherembodiment, the tetracycline responsive state is not a bacterialinfection. In another embodiment, the tetracycline compounds of theinvention are essentially non-antibacterial. For example,non-antibacterial tetracycline compounds of the invention may have MICvalues greater than about 4 μg/ml (as measured by assays known in theart and/or the assay given in Example 2).

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.)are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The inflammatory process is the response of living tissue todamage. The cause of inflammation may be due to physical damage,chemical substances, micro-organisms, tissue necrosis, cancer or otheragents. Acute inflammation is short-lasting, lasting only a few days. Ifit is longer lasting however, then it may be referred to as chronicinflammation.

IPAF's include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention.The disorders, diseases and states described in U.S. Pat. Nos.6,231,894; 6,015,804; 5,919,774; and 5,789,395 are also included as NOassociated states. The entire contents of each of these patents arehereby incorporated herein by reference.

Other examples of NO associated states include, but are not limited to,malaria, senescence, diabetes, vascular stroke, neurodegenerativedisorders (Alzheimer's disease & Huntington's disease), cardiac disease(reperfusion-associated injury following infarction), juvenile diabetes,inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute,recurrent and chronic infections (bacterial, viral and fungal); acuteand chronic bronchitis, sinusitis, and respiratory infections, includingthe common cold; acute and chronic gastroenteritis and colitis; acuteand chronic cystitis and urethritis; acute and chronic dermatitis; acuteand chronic conjunctivitis; acute and chronic serositis (pericarditis,peritonitis, synovitis, pleuritis and tendonitis); uremic pericarditis;acute and chronic cholecystis; cystic fibrosis, acute and chronicvaginitis; acute and chronic uveitis; drug reactions; insect bites;burns (thermal, chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states characterized by aberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention.

Examples of matrix metalloproteinase associated states (“MMPAS's”)include, but are not limited to, arteriosclerosis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis(Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8;238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

Examples of tetracycline responsive states also include neurologicaldisorders which include both neuropsychiatric and neurodegenerativedisorders, but are not limited to, such as Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amylotrophic lateral sclerosis (ALS), progressivesupranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomicfunction disorders such as hypertension and sleep disorders, andneuropsychiatric disorders, such as depression, schizophrenia,schizoaffective disorder, Korsakoff's psychosis, mania, anxietydisorders, or phobic disorders; learning or memory disorders, e.g.,amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affectiveneurological disorders, e.g., migraine and obesity. Further neurologicaldisorders include, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which is incorporated herein byreference in its entirety.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound,(e.g., inhibitor) and with the other therapeutic agent or treatment,administration of the tetracycline compound first, followed by the othertherapeutic agent or treatment and administration of the othertherapeutic agent or treatment first, followed by the tetracyclinecompound. The other therapeutic agent may be any agent which is known inthe art to treat, prevent, or reduce the symptoms of an IPAS.Furthermore, the other therapeutic agent may be any agent of benefit tothe patient when administered in combination with the administration ofan tetracycline compound. In one embodiment, the cancers treated bymethods of the invention include those described in U.S. Pat. Nos.6,100,248; 5,843,925; 5,837,696; or 5,668,122, incorporated herein byreference in their entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,or diabetes type II. In a further embodiment, protein glycosylation isnot affected by the administration of the tetracycline compounds of theinvention. In another embodiment, the tetracycline compound of theinvention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy. In a furtherembodiment, the IPAS includes disorders described in U.S. Pat. Nos.5,929,055; and 5,532,227, incorporated herein by reference in theirentirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders where asubjects bones are disorders and states where the formation, repair orremodeling of bone is advantageous. For examples bone mass disordersinclude osteoporosis (e.g., a decrease in bone strength and density),bone fractures, bone formation associated with surgical procedures(e.g., facial reconstruction), osteogenesis imperfecta (brittle bonedisease), hypophosphatasia, Paget's disease, fibrous dysplasia,osteopetrosis, myeloma bone disease, and the depletion of calcium inbone, such as that which is related to primary hyperparathyroidism. Bonemass disorders include all states in which the formation, repair orremodeling of bone is advantageous to the subject as well as all otherdisorders associated with the bones or skeletal system of a subjectwhich can be treated with the tetracycline compounds of the invention.In a further embodiment, the bone mass disorders include those describedin U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE 34,656;5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of which is herebyincorporated herein by reference in its entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Traumaincludes any injury to living tissue caused by an extrinsic agent orevent. Examples of trauma include, but are not limited to, crushinjuries, contact with a hard surface, or cutting or other damage to thelungs.

The invention also pertains to a method for treating acute lung injuryby administering a substituted tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, cystic fibrosis, and emphysema.In a further embodiment, the tetracycline compounds of the inventionused to treat acute and/or chronic lung disorders such as thosedescribed in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and5,773,430, each of which is hereby incorporated herein by reference inits entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, or ischemic stroke. The invention also pertains to amethod for treating ischemia, stroke, or ischemic stroke byadministering an effective amount of a substituted tetracycline compoundof the invention. In a further embodiment, the tetracycline compounds ofthe invention are used to treat such disorders as described in U.S. Pat.Nos. 6,231,894; 5,773,430; 5,919,775 or 5,789,395, incorporated hereinby reference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgellatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,83915,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the in vitro standard broth dilutionmethod described in Waitz, J. A., National Commission for ClinicalLaboratory Standards, Document M7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd)edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

Exemplification of the Invention EXAMPLE 1 Synthesis of 9-AlkylAminomethyl Minocycline

Minocycline hydrochloride (compound 2) was dissolved in methylsulfonicacid or hydrofluoric acid with methylsulfonic anhydride. N-hydroxymethylphthalimide was added to the reaction mixture. The mixture was stirredat 20-35 C until the reaction was complete. The acid solution was addedto an ice/water mixture and the triflic salt was readily precipitated,filtered and collected. The salt was re-dissolved in acetone and broughtto a neutral pH with base. The product was precipitated by the additionof water. The product was isolated as a mixture of the bis and trisalkylated product. The isolated material of this reaction was enrichedin the desired bis ratio (90%).

The solid was suspended in the EtOH. Aminolysis was carried out by usingmethylamine. A phthalamide by-product precipitated as the reactionprogressed and was removed by filtration. The light yellow solid productwas precipitated out by the addition of about 1.5 volumes oft-butylmethylether to the reaction mixture, and collected through asimple filtration that left many small impurities and methylaminereagent in the solution. Further purification of the compound wasperformed through re-slurrying with methanol.

Compound 4 as freebase was transferred to a hydrogenation vessel whichwas charged with methanol and aldehyde. An inactivated Pd/C catalyst wascharged and the vessel was pressurized with hydrogen gas. The reactionmixture was hydrogenated under hydrogen pressure around 30 Psi for about24 hours. When conversion of compound 4 to 1 was complete, the solutionwas filtered and washed through a Celite pad. At this point the reactionmixture contained very low β C-4 epimer, around 3-7%.

The product (1) was worked up and isolated selectively from itsimpurities. The pH of the solution was adjusted to about 4.5 withconcentrated HCl and the solution was washed with dichloromethane.Sulfites were added to the aqueous layer and the product was extractedwith dichloromethane at pH of about 7 to 8 to selectively recover thepreferred epimer product (e.g., α). The dichloromethane layers werecombined and concentrated, and 2 L of n-heptane was added to precipitatethe product. Further purification was obtained by repeating the work-upprocedure with or without t-butylmethyl ether to dissolve the crudeproduct.

EXAMPLE 2 Isolation and Purification of an 9-Alkyl AminomethylMinocycline Compound

Crude 9-(2′,2′-dimethylpropyl aminomethyl) minocycline freebase (40 g)was dissolved in 150 mL of buffer A (0.1% aqueous solution ofmethanesulfonic acid—

MSA) and the pH was adjusted to 2-3 with MSA.

The solution was filtered and injected into an HPLC and the product waseluted with an isocratic gradient of 94% buffer A and 6% acetonitrile.The product fraction collection was initiated when the product peak wasdetected. Each fraction was analyzed and an acceptance criterion ofgreater than 80% AUC of the main peak was used for the early productfractions. When combining fractions, the level of impurities andrelative concentration of the pooled fractions was factored into theselection criteria that meets the final product specifications. To theproduct fractions was added a 10% aqueous solution of sodium sulfiteequal to 10% of the original volume of the collected fractions.

A product fraction volume of 3.5 liters (including sodium sulfite) wascollected and the pH was adjusted to 4.0-4.5 using a solution of sodiumhydroxide. The aqueous solution was washed with 2 liters ofdichloromethane and the organic layer was separated and discarded.

The pH of the aqueous layer was adjusted to 7.5-8.5 using sodiumhydroxide and the product was extracted four times with 2.4 liters ofdichloromethane. The pH was readjusted to 7.5 to 8.5 with sodiumhydroxide, prior to each extraction.

The four dichloromethane layers were combined and concentrated to about200 ml, which was then added slowly (over a period of about 10 minutes)to a vigorously stirred n-heptane (2.5 L). The suspension was stirredfor about 10 minutes at room temperature and diluted slowly (over aperiod of 5 minutes) with n-heptane 1.5 L. The slurry was cooled to 0-5°C. and stirred for 1-2 hours. The suspended solid was filtered andwashed with 3×150 mL portions of n-heptane. The product was dried undervacuum at 40° C. for at least 24 hours until a constant weight wasachieved and the levels of all residual solvents were withinspecification. Approximately 13.6 g of 9-(2′,2′-dimethylpropylaminomethyl) minocycline freebase was isolated as a yellow solid.

The off-cuts were isolated in a similar manner and yielded 1.64 g.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

All patents, patent applications, and literature references cited hereinare hereby expressly incorporated by reference.

1. A method of synthesizing an aminoalkyl tetracycline compound,comprising: contacting a tetracycline compound with aN-hydroxymethyl-phthalimide in the presence of a water scavenger and anacid under appropriate conditions, such that an aminomethyl tetracyclineintermediate compound is formed.
 2. The method of claim 1, wherein thetetracycline compound is minocycline.
 3. The method of claim 1, whereinthe N-hydroxymethyl phthalimide is of the formula:

wherein R¹ and R² are each hydrogen, alkyl, halogen, alkenyl, alkynyl,aryl, cyano, amino, amidino, alkoxy, and acyl.
 4. The method of claim 1,wherein said appropriate conditions a reaction temperature of about20-25 C.
 5. The method of claim 1, wherein said water scavenger is anacid chloride or an acid anhydride.
 6. The method of claim 5, whereinsaid acid anhydride is methylsulfonic anhydride.
 7. The method of claim1, wherein said acid is triflic acid or methane sulfonic acid.
 8. Themethod of claim 1, wherein said aminomethyl tetracycline intermediate isno more than 10% tris-alkylated.
 9. The method of claim 1, wherein saidaminomethyl tetracycline intermediate is further dissolved in acetone.10. The method of claim 1, further comprising treating the aminomethyltetracycline intermediate with methylamine under second appropriateconditions.
 11. The method of claim 9, wherein said second appropriateconditions comprise a alkyl alcohol and an alkyl ether.
 12. The methodof claim 11, wherein said second appropriate conditions comprise aninert atmosphere.
 13. The method of claim 10, wherein after treating theaminomethyl tetracycline intermediate with methylamine under secondappropriate conditions, a second aminomethyl tetracycline intermediateis formed. 14-15. (canceled)
 16. A method for the synthesis of anaminoalkyl tetracycline compound, comprising: contacting a tetracyclinecompound with a N-hydroxymethyl-phthalimide in the presence of a waterscavenger and an acid under appropriate conditions to form anaminomethyl tetracycline intermediate compound; treating saidaminomethyl tetracycline intermediate compound with methylamine undersecond appropriate conditions to form a second aminomethyl tetracyclineintermediate; treating said second aminomethyl tetracycline intermediateunder appropriate hydrogenation conditions, such that anaminomethyltetracycline compound is formed. 17-29. (canceled)
 30. Amethod of purifying alkylaminomethyl minocycline compounds, comprising:injecting an aqueous low pH solution of said compound into an HPLC in apolar organic solvent gradient, and combining the product fractions,such that the alkylaminomethyl minocycline compound is purified. 31-44.(canceled)
 45. A method of removing hydrophobic impurities and oxidativedegradents from an alkylaminomethyl minocycline compound, comprisingdissolving said minocycline compounds in an aqueous solution of a pH of4.0-4.5, washing said aqueous solution with a non-polar organic solvent,and discarding said non-polar organic solvent and retaining said aqueoussolution, such that hydrophobic impurities and oxidative degradents areremoved from said alkylaminomethyl minocycline compound.
 46. (canceled)47. A method of removing β epimer and by products from analkylaminomethyl minocycline compound, comprising dissolving saidminocycline compounds in an aqueous solution of a pH of 7.5-8.5, washingsaid aqueous solution with a non-polar organic solvent, and discardingsaid aqueous solution and retaining said non-polar organic solution,such that β epimer and by products are removed from saidalkylaminomethyl minocycline compound. 48-53. (canceled)
 54. Apharmaceutical composition comprising a tetracycline compoundsynthesized or purified by the method of claim 1 and a pharmaceuticallyacceptable carrier.
 55. A method for treating a tetracycline responsivestate in a subject, comprising administering to said subject aneffective amount of an aminoalkyl tetracycline compound synthesized orpurified by the method of claim 1, such the tetracycline responsivestate is treated.
 56. A method for treating a tetracycline responsivestate in a subject, comprising administering to said subject aneffective amount of an alkylaminomethyl minocycline compound synthesizedor purified by the method of claim 30, such the tetracycline responsivestate is treated.